Oligonucleotides and methods for inhibiting cellular proliferation

ABSTRACT

The present invention relates to methods of treating disease-associated cellular proliferation using oligonucleotides. In particular, it relates to the use of oligonulceotides which are substantially complementary to interleukin-6 receptor mRNA sequences. In the form of pharmaceutical compositions, these oligonucleotides are suitable for administration to human subjects for the treatment of abnormal cellular proliferation due to such diseases as cancer, autoimmune disorders and viral infection.

This application is a continuation of application Ser. No. 08/486,408filed Jun. 7, 1995, now U.S. Pat. No. 5,716,846.

FIELD OF THE INVENTION

The present invention relates to methods for treating disease usingoligonucleotides which are effective inhibitors of cellularproliferation. In particular, it relates to methods for inhibitingdisease-associated cellular proliferation using therapeutic agentscomprising oligonucleotides which are substantially complementary tointerleukin-6 receptor mRNA sequences.

BACKGROUND OF THE INVENTION

Cellular growth, function, differentiation and development are regulatedby a variety of different mechanisms. Among the most importantregulators of cells are the receptor-specific proteins called"cytokines". These proteins bind to specific membrane-associatedreceptors which, in turn, transduce intracellular signals thatulitmately regulate the expression of critical genes and thereby controlmany cellular functions.

Interleukin-6 ("IL-6") is one of the most well characterized and studiedcytokines. It functions through the interaction with at least twotransmembrane glyprotein receptor molecules on the surface of targetcells (Taga, et al., J. Exp. Med. 166: 967-981 (1987); the interleukin-6receptor ("IL-6R") and the signal transducer, gp130 (Yamasaki, et al.,Science 241: 825-828 (1988); and Hibi et al., Cell 63: 1149-1157(1990)). Signal transduction by IL-6 involves the concerted action ofboth IL-6R and gp130. Initially, IL-6 binds to IL-6R with low affinity(Taga et al., Cell 58: 573-581 (1989)). This initial binding eventinduces the formation of a ternary complex consisting of two moleculesof gp130 dimerized with two IL-6/IL-6R ligand-receptor complexes. Inthis ternary complex, IL-6 is bound with high affinity resulting in thetransduction of intracellular signals by the gp130 molecules (Ward etal., J. Biol. Chem. 269: 23286-23289 (1994)).

In addition to playing an important role in modulating normal cellularfunction, IL-6 overproduction has been implicated in many differentdisease states. Recently, many investigators have focused on thesuppression of IL-6 production, function and/or signal transduction aspotentially useful means of inhibiting the cellular proliferation whichis associated with different disease states. Vink, et al. (J. Exp. Med.1: 997-1000(1990)) describe the inhibition of plasmacytoma growth invivo by using antibodies directed against IL-6 or its receptorcomponent, IL-6R. More recently, antisense oligonucleotides have beenstudied for use as inhibitors of cellular proliferation. Levy et al. (JClin. Invest. 88: 696-699 (1991)) describe the use of antisenseoligonucleotides which are complementary to the mRNA encoding the IL-6protein. Fujita (PCT Application No. WO 94/25036) describes the use ofantisense oligonucleotides which are complementary to the initiatorcodon of the mRNA encoding IL-6R.

The field of "antisense therapeutics" refers to the use ofoligonucleotides which are complementary to target nucleic acids, mostusually mRNA, as regulators of nucleic acid function. An antisenseoligonucleotide, i.e. an oligonucleotide having a nucleic acid sequencewhich is complementary to that of the "sense" nucleic acid to which itis targeted, can function in many different ways to modulate nucleicacid function. When the targeted nucleic acid is MRNA, it may functionby preventing translation of the MRNA into protein or inhibiting thebinding or translocation of ribosomes. When the targeted nucleic acid isDNA, it may prevent transcription into mRNA.

In addition to inhibiting the production and/or function of MRNA by a"sequence specific" antisense mechanism, the effect of certainoligonucleotides, and particularly phosphorothioate oligonucleotides,can be partially attributed to non-sequence specific mechanisms. Suchmechanisms have been reported to account for some of the effects ofphosphorothioate oligonucleotides as anti-viral agents. (Stein, et al.,Pharmac. Ther. 52: 365-384 (1991); Majumdar, et al., Biochemistry 28:1340 (1989)).

It is an object of the present invention to provide oligonucleotideswhich effectively inhibit disease-associated cellular proliferationand/or growth. Such oligonucleotides are complementary to the mRNAencoding the IL-6R, and function via sequence specific and/ornon-sequence specific mechanisms. A further objective of the presentinvention is to provide pharmaceutical compositions suitable foradministration to human subjects comprising these oligonucleotides.

SUMMARY OF THE INVENTION

The present invention features methods for inhibiting disease-associatedcellular proliferation using therapeutic agents comprisingoligonucleotides which are substantially complementary to interleukin-6receptor MRNA sequences. The preferred uses of the methods describedherein are in the treatment of a patient suffering from cancer, such asrenal cell carcinoma, an autoimmune disease or a viral infection. Otheruses of the present invention include detecting the presence of theinterleukin-6 receptor mRNA by using the oligonucleotides as in vitrodetection probes. These detection probes would be particularly useful inevaluating the effectiveness of other therapeutic agents in reducinginterleukin-6 receptor mRNA levels.

The method of the present invention employs therapeutic agents composedof oligonucleotides which are specific for interleukin-6 receptor mRNA.The preferred oligonucleoitdes are based on the following sequences:

    SEQ. ID. NO. 2                                                                             CCACAGCGCC GCACCTGAGC                                            SEQ. ID. NO. 3                                                                             GAAAACATTT GAGGAACTC                                             SEQ. ID. NO. 4                                                                             ACACTGCGAG TCCCTCG                                               SEQ. ID. NO. 5                                                                             GCGGACTGGC TAATGGGAA                                             SEQ. ID. NO. 6                                                                             GAGTCGTGGA GCTGCACCGA                                            SEQ. ID. NO. 7                                                                             GCTCCGAGGA CCCCACTCA                                             SEQ. ID. NO. 8                                                                             CGGGACTGCT AACTGGCA                                              SEQ. ID. NO. 9                                                                             GCTCCCGACA CTACTGGCGA C                                          SEQ. ID. NO. 10                                                                            GGTGGACACC TCGTTCT                                               SEQ. ID. NO. 11                                                                            TTTCCCCTGG CGTAGAACCT                                            SEQ. ID. NO. 12                                                                            GGGCAGCCAG CAGCGCGCA                                             SEQ. ID. NO. 13                                                                            AGCTGCACCG ACCTCAGCAG CAG                                        SEQ. ID. NO. 14                                                                            CCTGCTGCCG GCTTCCTGAG                                            SEQ. ID. NO. 15                                                                            TCTGCTGGGG TGGGAGCCTG CA                                         SEQ. ID. NO. 16                                                                            CCCATGCCAG CCCATCTCCT                                        

Oligonucleotides having nucleic acid sequences substantiallycorresponding to a preferred nucleic acid sequence and consistingessentially of the preferred nucleic acid sequence (i.e. having anucleic acid sequence which is substantially the same) are also coveredby the present invention. In particular, nucleic acid sequences whichsubstantially correspond to SEQ. ID. NO.s 5, 14 and 16 above, and aregiven by SEQ. ID. NO. 17, 18 and 19 as follows, respectively, areexpressly covered by the present invention:

    SEQ. ID. NO. 17                                                                            GCGGACAGGC TAATGGGAA                                             SEQ. ID. NO. 18                                                                            CCTGCAGCCG GCTTCCTGAG                                            SEQ. ID. NO. 19                                                                            CCCATGCCAG CCCATCTGCT                                        

More particularly, the present invention features a method of inhibitingdisease-associated cellular proliferation comprising the step ofcontacting the cells with a purified oligonucleotide 12 to 100nucleotides in length, said oligonucleotide being substantiallycomplementary to a nucleic acid sequence in the mRNA encoding theinterleukin-6 receptor, wherein said oligonucleotide consistsessentially of a nucleic acid sequence selected from the groupconsisting of:

    SEQ. ID. NO. 2                                                                             CCACAGCGCC GCACCTGAGC                                            SEQ. ID. NO. 3                                                                             GAAAACATTT GAGGAACTC                                             SEQ. ID. NO. 4                                                                             ACACTGCGAG TCCCTCG                                               SEQ. ID. NO. 5                                                                             GCGGACTGGC TAATGGGAA                                             SEQ. ID. NO. 6                                                                             GAGTCGTGGA GCTGCACCGA                                            SEQ. ID. NO. 7                                                                             GCTCCGAGGA CCCCACTCA                                             SEQ. ID. NO. 8                                                                             CGGGACTGCT AACTGGCA                                              SEQ. ID. NO. 9                                                                             GCTCCCGACA CTACTGGCGA C                                          SEQ. ID. NO. 10                                                                            GGTGGACACC TCGTTCT                                               SEQ. ID. NO. 11                                                                            TTTCCCCTGG CGTAGAACCT                                            SEQ. ID. NO. 12                                                                            GGGCAGCCAG CAGCGCGCA                                             SEQ. ID. NO. 13                                                                            AGCTGCACCG ACCTCAGCAG CAG                                        SEQ. ID. NO. 14                                                                            CCTGCTGCCG GCTTCCTGAG                                            SEQ. ID. NO. 15                                                                            TCTGCTGGGG TGGGAGCCTG CA                                         SEQ. ID. NO. 16                                                                            CCCATGCCAG CCCATCTCCT                                            SEQ. ID. NO. 17                                                                            GCGGACAGGC TAATGGGAA                                             SEQ. ID. NO. 18                                                                            CCTGCAGCCG GCTTCCTGAG                                            SEQ. ID. NO. 19                                                                            CCCATGCCAG CCCATCTGCT                                        

Yet another aspect of the present invention is a method of inhibitingdisease-associated proliferation of cells comprising the step ofcontacting the cells with a purified oligonucleotide 12 to 100nucleotides in length, said oligonucleotide being substantiallycomplementary to a nucleic acid sequence region of 50 nucleotidespresent in the mRNA encoding IL-6R (abut having T substituted for U)consisting of:

    SEQ. ID. NO. 1:                                                               CGGCTGCAGG CTCCCACCCC AGCAGATGGG CTGGCATGGG                                   AAGGAGGCTG,                                                               

wherein said oligonucleotide inhibits proliferation of cells in vivo orin vitro.

Other features and advantages of the invention are apparent from thefollowing detailed description and the claims.

DETAILED DESCRIPTION

The present invention concerns methods of using oligonucleotides whichinhibit cellular proliferation. In order to more clearly describe thesubject matter of the present invention, certain terms used herein shallbe defined as follows unless otherwise indicated:

Antisense Oligonucleotide: "Antisense oligonucleotide" means anoligonucleotide which is complementary to a target "sense" nucleic acid,and functions at least partially by sequence-specific mechanisms toregulate the functioning of the target nucleic acid.

Complementary: "Complementary", when used to refer to a nucleic acid,means a nucleic acid of one polarity containing a sequence ofnucleotides whose bases pair via Watson-Crick hydrogen bonds with thenucleotide bases of another nucleic acid of opposite polarity, i.e.adenine ("A") pairs with thymine ("T") or uracil ("U"), and guanine("G") pairs with cytosine ("C"). For example, a nucleic acid having thesequence GCAU in the 5' to 3' direction is "complementary" to a nucleicacid having the sequence CGTA in the 3' to 5' direction. Use of the termcomplementary herein is intended to include those nucleic acids whichare substantially complementary such that, despite occasional mismatchesbetween the strands, a stable duplex will nevertheless be formed. Theindividual strands of a complementary nucleic acid pair can also bereferred to as the plus ("(+)") or "sense" strand and the minus ("(-)")or "antisense" strand.

Disease-associated Cellular Proliferation: "Disease-associated cellularproliferation" means an abnormal level of cell division and/or growthwhich is caused by or associated with a particular disease such ascancer or viral infection.

Hybridize: "Hybridize" means the formation of a duplex betweencomplementary nucleic acids via base pair interactions.

Liposome: "Liposome" means a vesicle composed of amphipathic lipidsarranged in a spherical bilayer or bilayers.

Modified: "Modified", when used to refer to a nucleic acid, means anucleic acid in which any of the natural structures have been altered.These include modifications to the phosphodiester linkages, the sugars(ribose in the case of RNA or deoxyribose in the case of DNA) and/or thepurine or pyrimidine bases. Modified phosphodiester linkages includephosphorothioates, phosphotriesters, methylphosphonates andphosphorodithioates. "Modified dNTPs" refers to nucleoside triphosphateswhich, when incorporated into a nucleic acid, will result in theformation of modified nucleic acids.

Nucleic Acid Sequence: "Nucleic acid sequence", or "sequence", meansboth a nucleic acid having a particular sequence of nucleotides, andalso the sequence or order of nucleotides present in a particularnucleic acid. Which of these two meanings applies will be apparent formthe context in which this term is used.

Oligonucleotide: "Oligonucleotide" means an oligodeoxyribonucleotidehaving a defined nucleic acid sequence.

Pharmacologically compatible carrier: "Pharmacologically compatiblecarrier means a formulation to which the oligonucleotide can be added tofacilitate its administration to a patient and/or efficacy withoutexhibiting any unacceptable levels of toxicity or pharmacologicallyadverse effects.

Phosphorothioate oligonucleotide: "Phosphorothioate oligonucleotide"means an oligonucleotide having all phosphorothioate linkages in placeof naturally occurring phosphodiester linkages.

Phosphorothioate-containing oligonucleotide:"Phosphorothioate-containing oligonucleotide" means an oligonucleotidehaving at least one and as many as all phosphorothioate linkages. Thisterm is intended to include phosphorothioate oligonucleotides.

Polarity: "Polarity" means the orientation of a nucleic acid polymerwhich is created when the C3 position of one deoxyribose (or ribose)moiety is linked together with the C5 of the adjacent deoxyribose (orribose) moiety via a phosphate linkage. The polarity of nucleic acids isreferred to as 5' to 3' or 3' to 5'.

Polymerase: "Polymerase" means an enzyme which is capable of catalyzingthe sequential addition of nucleotides to a nucleic acid.

Primer: "Primer" means an oligonucleotide that is complementary to atemplate that hybridizes with the template to initiate synthesis by apolymerase, such as reverse transcriptase, and which is extended by thesequential addition of covalently bonded nucleotides linked to its 3'end that are complementary to the template.

Substantially Complementary: "Substantially Complementary", when used torefer to a nucleic acid, means having a sequence such that not all ofthe nucleotides exhibit base pairing with the nucleotides of anothernucleic acid, but the two nucleic acids are nonetheless capable offorming a stable hybrid under appropriate conditions.

Template: "Template" means the nucleic acid having the sequence ofnucleotides which will provide the pattern and serve as substrate forproducing a desired oligonucleotide. In order to serve as such, thetemplate must also contain a sequence which is capable of hybridizingwith a primer or, in the case of self-priming templates, capable offorming a self-priming region.

Therapeutically effective amount: "Therapeutically effective amount"means an amount which is effective in inhibiting disease-associatedcellular proliferation and/or growth in a patient suffering from adisease associated with overproduction of IL-6. Preferably, thetherapeutically effective amount relieves, to some extent one or moresymptoms associated with the disease.

The development of therapeutic applications using oligonucleotides isnow widespread. Although the precise mechanism of action ofoligonucleotides as therapeutic agents is often difficult to determine,many proposed mechanisms have been suggested and any or all of thesedifferent mechanisms may act in concert to produce a desired result. Onemechanism of action is based on antisense. Antisense oligonucleotidesare generally designed to have sequences which are complementary tospecific sequences found in target nucleic acids such as DNA, mRNA orprecursor mRNA. By hybridizing to a specific sequence in the targetnucleic acid, the antisense oligonucleotide interrupts theprotein-encoding function of the DNA.

Some of the proposed mechanisms which may account for the antisensefunction of a particular oligonucleotide may include: cleavage of RNA ina RNA:DNA hybrid by an enzyme having RNase H activity; prematuretermination of mRNA transcription; prevention of translocation of themRNA to the site for protein translation; interference with mRNAprocessing by hybridizing to an mRNA intron/exon; interference with mRNAfunction by hybridizing to non-protein coding (untranslated) regions;and/or interference with ribosome binding by hybridizing to an mRNAinitiator codon. In summary, each of these sequence-specific antisensemechanisms act in some way to inhibit the expression of a particulargene.

In addition to sequence-specific antisense mechanisms, certain modifiedoligonucleotides can inhibit nucleic acid function via a non-sequencespecific mechanism. In some instances, when the effects of antisenseoligonucleotides are compared to "control" oligonucleotides whichcontain the same bases in randomized order, the control oligonucleotidesalso exhibit inhibition of protein production. Although the precisemechanism for such non-sequence specific mechanisms is not known, theseeffects have been attributed to the accidental inhibition of otheressential genes by the control oligonucleotides. (See Milligan, et al.,in Antisense Therapeutics; Development of Antisense Therapeutics, Annalsof the New York Academy of Sciences, p. 229-241.)

One potential explanation for non-sequence specific effects ofoligonucleotides on proliferation of renal carcinoma cells is theinhibition of topoisomerase. Many anticancer agents with activityagainst renal cell carcinoma have been demonstrated to inhibittopoisomerase. (Shuin, et al., Anticancer Research 14: 2621-2626(1994)). It is hypothesized that topoisomerase inhibition byphosphorothioate oligonucleotides may account for a portion of theobserved antiproliferative effects attributable to the phosphorothioateoligonucleotide.

In any case, both sequence specific and non-sequence specific mechanismsmay account for the effects of the oligonucleotides of the presentinvention. A complete understanding of the mechanisms of action is notnecessary for the design of cellular function-inhibitingoligonucleotides.

The oligonucleotides of the present invention are complementary to themRNA encoding the IL-6R protein, and may inhibit IL-6R production viaantisense and/or other mechanisms. Therapeutic agents which inhibit thefunctioning of IL-6, such as those which regulate the production ofIL-6R, can be used to counteract the effects of overproduction of IL-6.The normal functioning of IL-6 involves the induction of IL-6 productionby many different cell types, such as fibroblasts, macrophages,endothelial cells and keratinocytes, as a response to injury orinfection. In the absence of injury or infection, these cells do notnormally produce IL-6. IL-6 production results in an enhancement of theimmune response via a variety of mechanisms which include B- and T- cellproliferation or differentiation as well as T-cell and macrophageactivation.

However, IL-6 overproduction is implicated in many different diseasestates. For example, IL-6 hyperexpression in Epstein Barr virus infectedB lymphocytes has been shown to be partially responsible fortumorigenicity (Scala et al., J. Exp. Med. 17: 61-68 (1990)). Theoverproduction of IL-6 by keratinocytes has been shown to play acausative role in the epidermal hyperplasia associated with psoriasis(Grossman et al., Proc. Nat. Acad. Sci. 86: 6367-6371(1989)).Overproduction of IL-6 by renal carcinoma cells has been shown to beassociated with increased metastases (Takenawa, et al., Journal of theNational Cancer Institute 83(22): 1668-1672(1991)). IL-6 also plays areported role in the increase of bone resorption during menopause due toits enhancement of osteoclast development (Jilka et al., Science 257:88-91 (1992); and Girasole et al., Journal of Clinical Investigation 89:883-891(1992)). Additionally, IL-6 has been shown to be a tumor growthfactor for multiple myeloma cells (Klein, et al., Eur. Cytokine Net.,1(4): 193-201(1990)). Other disease states which have been associatedwith IL-6 overproduction include plasma cell leukemia, cachexia,mesangial proliferative glomerulonephritis, Kaposi's sarcoma, rheumatoidarthritis, hypergammaglobulinemia, Castleman's disease, IgM gamopathy,cardiac myxoma and autoimmune insulin-dependent diabetes.

Thus, therapeutic agents which are designed to inhibit IL-6 functionhave a widespread therapeutic application. They can be used as atherapeutic agent in the treatment of any of the aforementioned diseasestates. The antisense oligonucleotides of the present invention arepreferably used to treat renal cell carcinoma.

The oligonucleotides of the present invention can be either DNA or RNA,but are preferably DNA. The oligonucleotides can be prepared using anyknown chemical or enzymatic methods. Chemical synthesis can beconveniently performed according to the method described by Stec et al.(J. Am. Chem. Soc. 106: 6077-6079 (1984)) using the phosphoroamiditemethod and an automated synthesizer, such as Model 380-B (AppliedBiosystems, Inc., Foster City, Calif.).

The oligonucleotides included within the present invention can be eitherunmodified or modified. Modified oligonucleotides can be prepared byaltering any of the natural structures of a nucleic acid. Thesestructures include the phosphodiester linkages, the sugars (ribose inthe case of RNA or deoxyribose in the case of DNA) and/or the purine orpyrimidine bases. Any modification can be made to an oligonucleotide aslong as it does not render the oligonucleotide ineffective athybridizing to the target nucleic acid or toxic, if to be used in vivo.This includes certain modifications which may diminish hybridizationefficiency without completely preventing the formation of a stableduplex.

Preferred modifications are to the phosphodiester linkages to renderthem more stable in the presence of nucleases. Modifying thephosphodiester linkages may also enhance cellular uptake. Modifiedphosphodiester linkages include phosphorothioate, methylphosphonate,phosphorodithioate, or phosphoselenate linkages. The oligonucleotidesmay contain all modified linkages, a mixture of different modifiedlinkages, a mixture of modified linkages and unmodified linkages, or anycombination of these which are either selectively positioned, or presentin different regions of the oligonucleotide as in a chimericoligonucleotide. Oligonucleotides with modified internucleotide linkagescan be synthesized in the same manner as unmodified oligonucleotides byknown methods, including many of the methods discussed above.

Other examples of modifications include the incorporation of modifiedsugar groups such as alpha-anomers or the sugars incorporated into2'-O-methyloligonucleotides. Also contemplated are modifications to thenucleotide purine or pyrimidine bases.

Preferably, the oligonucleotides of the present invention containphosphorothioate linkages which increase stability, facilitate cellularuptake and may enable the oligonucleotides to inhibit cellular functionsby sequence independent mechanisms as well as sequence specificantisense mechanisms.

The antisense oligonucleotides of the present invention are preferablyabout 12 to 100 nucleotides in length. More preferably, theseoligonucleotides are about 14 to 50 nucleotides in length, and mostpreferably about 18 to 35 nucleotides in length. Oligonucleotide lengthshould be selected to optimize the efficiency of the oligonucleotide ininhibiting disease-associated cellular proliferation and/or growth. Theexistence of any modifications in the oligonucleotide will alsoinfluence the effects of length on overall efficiency of theoligonucleotide.

In order to determine the optimal oligonucleotide size, several factorsshould be taken into account. Oligonucleotides which are short have theadvantage of being more easily internalized by cells. However, if theyare not long enough, for example less than 10 bases, they may not formspecific and stable hybrids with target sequence. On the other hand,longer oligonucleotides may hybridize to their targets with increasedstability which may enhance translation arrest by preventing a ribosomefrom displacing the oligonucleotide. However, if the oligonucleotide istoo long, for example greater than 150 bases, it may not be efficientlytaken up by cells and/or could potentially be cytotoxic.

An oligonucleotide screening assay designed to mimic physiologicalconditions can be utilized to predict the efficiency with which theoligonucleotides hybridize in living cells. Such a screening assay isdescribed by Nelson et al., in WO 95/03427.

The oligonucleotides of the present invention are specific for aparticular target sequence. More particularly, this target sequence isthe mRNA which encodes IL-6R and is described by Yamasaki, et al.,Science 241: 825-828 (1988). By being "specific for", it is intendedthat the oligonucleotides are complementary to the target sequence.Preferably, the target sequence is an early protein coding region of theIL-6R mRNA given by SEQ. ID. NO. 1 as follows:

    SEQ. ID. NO. 1:                                                               CGGCTGCAGG CTCCCACCCC AGCAGATGGG CTGGCATGGG AAGGAGGCTG                    

Preferred oligonucleotides of the present invention are given by thefollowing sequences:

    SEQ. ID. NO. 2                                                                             CCACAGCGCC GCACCTGAGC                                            SEQ. ID. NO. 3                                                                             GAAAACATTT GAGGAACTC                                             SEQ. ID. NO. 4                                                                             ACACTGCGAG TCCCTCG                                               SEQ. ID. NO. 5                                                                             GCGGACTGGC TAATGGGAA                                             SEQ. ID. NO. 6                                                                             GAGTCGTGGA GCTGCACCGA                                            SEQ. ID. NO. 7                                                                             GCTCCGAGGA CCCCACTCA                                             SEQ. ID. NO. 8                                                                             CGGGACTGCT AACTGGCA                                              SEQ. ID. NO. 9                                                                             GCTCCCGACA CTACTGGCGA C                                          SEQ. ID. NO. 10                                                                            GGTGGACACC TCGTTCT                                               SEQ. ID. NO. 11                                                                            TTTCCCCTGG CGTAGAACCT                                            SEQ. ID. NO. 12                                                                            GGGCAGCCAG CAGCGCGCA                                             SEQ. ID. NO. 13                                                                            AGCTGCACCG ACCTCAGCAG CAG                                        SEQ. ID. NO. 14                                                                            CCTGCTGCCG GCTTCCTGAG                                            SEQ. ID. NO. 15                                                                            TCTGCTGGGG TGGGAGCCTG CA                                         SEQ. ID. NO. 16                                                                            CCCATGCCAG CCCATCTCCT                                            SEQ. ID. NO. 17                                                                            GCGGACAGGC TAATGGGAA                                             SEQ. ID. NO. 18                                                                            CCTGCAGCCG GCTTCCTGAG                                            SEQ. ID. NO. 19                                                                            CCCATGCCAG CCCATCTGCT                                        

Particularly preferred sequences are given by SEQ. ID. NO.s 15 and 16.

It is not necessary for the entire oligonucleotide sequence to beperfectly complementary to the target IL-6R mRNA sequence. It is onlynecessary for the oligonucleotide to be "substantially complementary",i.e. capable of forming a stable hybrid with the target. Additionalnon-complementary nucleotides may be present in the antisenseoligonucleotide at any location, for example at either the 3' or 5'terminus, or any other location therebetween. Such additionalnon-complementary nucleotides may serve to inhibit in-vivo degradationand/or enhance the effects of the oligonucleotide in interfering withgene expression. The amount of complementarity necessary to form astable hybrid with the target sequence will depend on the types andamounts of modifications present, the types of bases involved inhydrogen bonding (e.g. G:C hydrogen bonding is stronger than A:T) andthe length of the oligonucleotide.

In addition to their usefulness as therapeutic agents, theoligonucleotides described herein are also useful as diagnostic probesand as research tools, such as amplification primers. Utilizing labeledoligonucleotide probes which are specific for IL-6R mRNA, the presenceor amount of IL-6R mRNA can be determined. The design and production oflabeled oligonucleotide probes and their use in hybridization methods iseasily accomplished by one of skill in the art.

Considerations for therapeutic use include oligonucleotide pharmacologyand delivery. For use as therapeutic agents, the oligonucleotides mustbe pharmacologically suitable, i.e. they must exhibit minimal toxicityand suitable distribution and metabolism. Different pharmacologicalconsiderations can be evaluated using techniques which are known in theart.

Pharmaceutical compositions comprising the oligonucleotides in apharmacologically acceptable carrier may be administered by a variety ofdifferent mechanisms which are well known to those of skill in the art.Such mechanisms include oral administration (inhalation or parenteral),injection (intravenous, intramuscular, subcutaneous, intraperitoneal),and topical administration (intranasally, cutaneous). Compositions whichare suitable for each of these different mechanisms are routinelyprepared and utilized.

Examples of pharmacologically acceptable carriers include aqueoussolutions such as water, saline, buffers or carbohydrate solutions; anddelivery vehicles such as liposomes, microspheres, or emulsions.Delivery vehicles can be utilized to enhance in vivo stability.Liposomes are preferred because of their ability to enhanceintracellular delivery, their long circulation half-lifes, the ease ofincorporation of receptor targeted molecules, their minimal toxicity andgood biodegradability. Liposomes may be made by a variety of techniquesknown in the art. (See, for example, Bangham et al., J. Mol. Biol., 13:238-252(1965)). These methods generally involve first dissolving andmixing the lipids in an organic solvent, followed by evaporation. Thenan appropriate amount of the aqueous phase is mixed with the lipidphase, and then allowed to incubate for a sufficient time for theliposomes to form. The aqueous phase will generally consist of thebiomolecule in suspension with other solutes, such as buffers or sugars.

The exact dosage and number of doses of the pharmaceutical compositionsdescribed herein depends upon several factors such as the diseaseindication, the route of administration, the delivery vehicle and theoligonucleotide composition. Duration of treatment will depend on theeffects of the treatment on the disease symptoms, and may includemultiple daily doses for extended periods of time.

EXAMPLE I Synthesis of Oligoncleotides

Oligonucleotides containing phosphodiester linkages as well as modifiedlinkages such as phosphorothioates can be synthesized by procedures wellknown in the art. For example, in Methods in Enzymology 154:287 (1987),Caruthers et al. describe a procedure for synthesizing oligonucleotidescontaining phosphodiester linkages by standard phosphoramiditesolid-phase chemistry. Bhatt, U.S. Pat. No. 5,253,723, describes aprocedure for synthesizing oligonucleotides containing phosphorothioatelinkages. Klem et al., PCT W092/07864 describe the synthesis ofoligonucleotides having different linkages including methylphosphonatelinkages.

Example II Inhibition of Renal Cell Carcinoma Cellular Proliferation byPhosphorothioate Oligonucleotides

In order to test the effectiveness of several different phosphorothioateoligonucleotides complementary to mRNA for IL-6R as inhibitors of cancercell proliferation, two different cell lines were studied. Caki-1 cells(American Type Culture Collection, Rockville, Md.), a renal cellcarcinoma derived cell line known to produce abnormally high levels ofIL-6 were used, with 293 cells (American Type Culture Collection,Rockville, Md.), an EBV transformed normal renal cell line serving asthe control.

Caki-1 or 293 cells were cultured in 48 well plates under conditionswhere the cells did not become confluent within the experimental timecourse. After the cells adhered to the plates (4-6 hours), cell culturemedium containing the phosphorothioate oligonucleotides was added to thecultures, and medium alone was added to control cells. The cells wereincubated under standard conditions (37° C., 5% CO₂), and on day 4 themedium was replaced. On day 7, the medium was removed and the cellsreleased from the plates using trypsin. Cell numbers per well weredetermined by counting cell density using a hemocytometer. As shown inTable 2, 1 μM antisense oligonucleotides inhibited cell proliferation ofCaki-1 cells by 70-90% but had little effect on the proliferation ofcontrol cells.

                  TABLE II                                                        ______________________________________                                        INHIBITION OF CELLULAR PROLIFERATION                                          BY PHOSPHOROTHIOATE OLIGONUCLEOTIDES                                                         % Reduction of                                                 Oligonucleotide                                                                              Caki-1 Cells                                                   ______________________________________                                        SEQ. ID. NO. 2 57                                                             SEQ. ID. NO. 3 38                                                             SEQ. ID. NO. 4 33                                                             SEQ. ID. NO. 5 45                                                             SEQ. ID. NO. 6 33                                                             SEQ. ID. NO. 7 42                                                             SEQ. ID. NO. 8 33                                                             SEQ. ID. NO. 9 52                                                             SEQ. ID. NO. 10                                                                              41                                                             SEQ. ID. NO. 11                                                                              35                                                             SEQ. ID. NO. 12                                                                              66                                                             SEQ. ID. NO. 13                                                                              43                                                             SEQ. ID. NO. 14                                                                              54                                                             SEQ. ID. NO. 15                                                                              92                                                             SEQ. ID. NO. 16                                                                              82                                                             ______________________________________                                    

Example III Inhibition of Multiple Myeloma Cell Proliferation byPhosphorothioate Oligonucleotides

Cellular proliferation in the presence of a phosphqrothioateoligonucleotide given by SEQ. ID. NO. 15 was performed as described inExample II using two different multiple myeloma cell lines; U266(American Type Culture Collection, Rockville, Md.) and RPMI (AmericanType Culture Collection, Rockville, Md.). Compared to theno-oligonucleotide control, there was a 34% and 20% reduction incellular proliferation in U266 and RPMI cells, respectively.

Although the invention is described in terms of specific embodiments,many modifications and variations of the present invention are possiblein light of the teachings. It is, therefore, to be understood thatwithin the scope of the appended claims the invention may be practicedotherwise than as specifically described.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 19                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #              50CACCCC AGCAGATGGG CTGGCATGGG AAGGAGGCTG                      - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 # 20               GAGC                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 # 19               CTC                                                        - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 # 19               GAA                                                        - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 # 20               CCGA                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 # 19               TCA                                                        - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #  18              CA                                                         - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #21                GCGA C                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #   17             T                                                          - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 20               ACCT                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                # 19               GCA                                                        - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #                23GCAG CAG                                                   - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                # 20               TGAG                                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #                 22CTG CA                                                    - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                # 20               TCCT                                                       - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                # 19               GAA                                                        - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                # 20               TGAG                                                       - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                # 20               TGCT                                                       __________________________________________________________________________

We claim:
 1. An oligonucleotide up to 35 nucleotide bases in length and comprising at least 18 contiguous nucleotide bases which are perfectly complementary to a nucleotide base sequence region contained in a nucleic acid sequence consisting of:

    SEQ ID NO. 1                                                                              CGGCTGCAGG CTCCCACCCC AGCAGATGGG                                               CTGGCATGGG AAGGAGGCTG.                                         


2. An oligonucleotide up to 35 nucleotide bases in length and comprising a nucleotide base sequence selected from the group consisting of:

    SEQ. ID. NO. 2                                                                              CCACAGCGCC GCACCTGAGC,                                            SEQ. ID. NO. 3                                                                              GAAAACATTT GAGGAACTC,                                             SEQ. ID. NO. 4                                                                              ACACTGCGAG TCCCTCG,                                               SEQ. ID. NO. 5                                                                              GCGGACTGGC TAATGGGAA,                                             SEQ. ID. NO. 6                                                                              GAGTCGTGGA GCTGCACCGA,                                            SEQ. ID. NO. 7                                                                              GCTCCGAGGA CCCCACTCA,                                             SEQ. ID. NO. 8                                                                              CGGGACTGCT AACTGGCA,                                              SEQ. ID. NO. 9                                                                              GCTCCCGACA CTACTGGCGA C,                                          SEQ. ID. NO. 10                                                                             GGTGGACACC TCGTTCT,                                               SEQ. ID. NO. 11                                                                             TTTCCCCTGG CGTAGAACCT,                                            SEQ. ID. NO. 12                                                                             GGGCAGCCAG CAGCGCGCA,                                             SEQ. ID. NO. 13                                                                             AGCTGCACCG ACCTCAGCAG CAG,                                        SEQ. ID. NO. 14                                                                             CCTGCTGCCG GCTTCCTGAG,                                            SEQ. ID. NO. 15                                                                             TCTGCTGGGG TGGGAGCCTG CA,                                         SEQ. ID. NO. 16                                                                             CCCATGCCAG CCCATCTCCT,                                            SEQ. ID. NO. 17                                                                             GCGGACAGGC TAATGGGAA,                                             SEQ. ID. NO. 18                                                                             CCTGCAGCCG GCTTCCTGAG, and                                        SEQ. ID. NO. 19                                                                             CCCATGCCAG CCCATCTGCT.                                       


3. The oligonucleotide of claim 1 or 2, wherein said oligonucleotide contains one or more modified sugars, one or more modified internucleoside linkages, or one or more modified sugars and one or more modified internucleoside linkages.
 4. The oligonucleotide of claim 3, wherein said modified internucleoside linkages are selected from the group consisting of phosphorothioate, methylphosphonate, phosphotriester, phosphorodithioate and phosphoselenate linkages.
 5. The oligonucleotide of claim 4, wherein said modified internucleoside linkages are phosphorothioate linkages.
 6. The oligonucleotide of claim 1 or 2, wherein said oligonucleotide is a phosphorothioate oligonucleotide.
 7. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 2  CCACAGCGCC GCACCTGAGC.                                    


8. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 3  GAAAACATTT GAGGAACTC.                                     


9. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 4  ACACTGCGAG TCCCTCG.                                       


10. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 5  GCGGACTGGC TAATGGGAA.                                     


11. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 6  GAGTCGTGGA GCTGCACCGA.                                    


12. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 7  GCTCCGAGGA CCCCACTCA.                                     


13. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 8  CGGGACTGCT AACTGGCA.                                      


14. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 9  GCTCCCGACA CTACTGGCGA C.                                  


15. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 10  GGTGGACACC TCGTTCT.                                      


16. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 11  TTTCCCCTGG CGTAGAACCT.                                   


17. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 12  GGGCAGCCAG CAGCGCGCA.                                    


18. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 13  AGCTGCACCG ACCTCAGCAG CAG.                               


19. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 14  CCTGCTGCCG GCTTCCTGAG.                                   


20. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 17  GCGGACAGGC TAATGGGAA.                                    


21. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 18  CCTGCAGCCG GCTTCCTGAG.                                   


22. The oligonucleotide of claim 2, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 19  CCCATGCCAG CCCATCTGCT.                                   


23. A method for inhibiting cytokine-induced cellular proliferation of a cell in culture, said method comprising the steps of:a) providing an oligonucleotide up to 35 nucleotide bases in length and comprising at least 18 contiguous nucleotide bases which are perfectly complementary to a nucleotide base sequence region contained in a nucleic acid sequence consisting of:

    SEQ ID NO. 1                                                                              CGGCTGCAGG CTCCCACCCC AGCAGATGGG                                               CTGGCATGGG AAGGAGGCTG; and                                     

b) contacting said cell with said oligonucleotide under conditions such that said oligonucleotide is delivered within said cell and hybridizes with said nucleotide base sequence region, thereby inhibiting cytokine-induced cellular proliferation of said cell.
 24. A method for inhibiting cytokine-induced cellular proliferation of a cell in culture, said method comprising the steps of:a) providing an oligonucleotide up to 35 nucleotide bases in and comprising a nucleotide base sequence selected from the group consisting of:

    SEQ. ID. NO. 2                                                                              CCACAGCGCC GCACCTGAGC,                                            SEQ. ID. NO. 3                                                                              GAAAACATTT GAGGAACTC,                                             SEQ. ID. NO. 4                                                                              ACACTGCGAG TCCCTCG,                                               SEQ. ID. NO. 5                                                                              GCGGACTGGC TAATGGGAA,                                             SEQ. ID. NO. 6                                                                              GAGTCGTGGA GCTGCACCGA,                                            SEQ. ID. NO. 7                                                                              GCTCCGAGGA CCCCACTCA,                                             SEQ. ID. NO. 8                                                                              CGGGACTGCT AACTGGCA,                                              SEQ. ID. NO. 9                                                                              GCTCCCGACA CTACTGGCGA C,                                          SEQ. ID. NO. 10                                                                             GGTGGACACC TCGTTCT,                                               SEQ. ID. NO. 11                                                                             TTTCCCCTGG CGTAGAACCT,                                            SEQ. ID. NO. 12                                                                             GGGCAGCCAG CAGCGCGCA,                                             SEQ. ID. NO. 13                                                                             AGCTGCACCG ACCTCAGCAG CAG,                                        SEQ. ID. NO. 14                                                                             CCTGCTGCCG GCTTCCTGAG,                                            SEQ. ID. NO. 15                                                                             TCTGCTGGGG TGGGAGCCTG CA,                                         SEQ. ID. NO. 16                                                                             CCCATGCCAG CCCATCTCCT,                                            SEQ. ID. NO. 17                                                                             GCGGACAGGC TAATGGGAA,                                             SEQ. ID. NO. 18                                                                             CCTGCAGCCG GCTTCCTGAG, and                                        SEQ. ID. NO. 19                                                                             CCCATGCCAG CCCATCTGCT; and                                   

b) contacting said cell with said oligonucleotide under conditions such that said oligonucleotide is delivered within said cell and hybridizes with a nucleic acid sequence encoding the IL-6 receptor of said cell, so that cytokine-induced cellular proliferation of said cell is inhibited.
 25. The method of claim 23 or 24, wherein said oligonucleotide contains one or more modified sugars, one or more modified internucleoside linkages, or one or more modified sugars and one or more modified internucleoside linkages.
 26. The method of claim 25, wherein said modified internucleoside linkages are selected from the group consisting of phosphorothioate, methylphosphonate, phosphotriester, phosphorodithioate and phosphoselenate linkages.
 27. The method of claim 23, wherein said modified internucleoside linkages are phosphorothioate linkages.
 28. The method of claim 24 or 26, wherein said oligonucleotide is a phosphorothioate oligonucleotide.
 29. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 2  CCACAGCGCC GCACCTGAGC.                                    


30. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 3  GAAAACATTT GAGGAACTC.                                     


31. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 4  ACACTGCGAG TCCCTCG.                                       


32. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 5  GCGGACTGGC TAATGGGAA.                                     


33. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 6  GAGTCGTGGA GCTGCACCGA.                                    


34. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 7  GCTCCGAGGA CCCCACTCA.                                     


35. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 8  CGGGACTGCT AACTGGCA.                                      


36. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 9  GCTCCCGACA CTACTGGCGA C.                                  


37. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 10  GGTGGACACC TCGTTCT.                                      


38. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 11  TTTCCCCTGG CGTAGAACCT.                                   


39. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 12  GGGCAGCCAG CAGCGCGCA.                                    


40. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 13  AGCTGCACCG ACCTCAGCAG CAG.                               


41. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 14  CCTGCTGCCG GCTTCCTGAG.                                   


42. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 17  GCGGACAGGC TAATGGGAA.                                    


43. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 18  CCTGCAGCCG GCTTCCTGAG.                                   


44. The method of claim 24, wherein said oligonucleotide consists of:

    SEQ. ID. NO. 19  CCCATGCCAG CCCATCTGCT.                                    